Application of CB-839 in preparation of medicine for treating diabetic kidney disease

Abstract

The invention discloses an application of a small molecule compound CB-839 in preparation of a medicine for treating diabetic kidney diseases. The treatment of the diabetic kidney disease means that the renal function and the kidney injury of the diabetic kidney disease are obviously improved by the CB-839 (C26H24F3N7O3S). The CB-839 is a glutaminase 1 (GLS1) inhibitor, and can prevent in-vivo glutamine decomposition by inhibiting the activity of glutamine decomposition enzyme so as to maintain the in-vivo concentration of the CB-839. In addition, the CB-839 shows antitumor activity in animal experiments. Experimental results show that the CB-839 can inhibit glutamine decomposition, so that the purpose of improving prognosis of the diabetic kidney disease is achieved.

Description

Technical Field

[0001] The present invention belongs to the technical field of disease treatment, and particularly relates to the application of a small molecule compound CB-839 in the preparation of a drug for treating diabetic kidney disease. Background Art

[0002] Diabetic kidney disease is one of the important microvascular complications of diabetes. Approximately 20% - 40% of diabetic patients in China are complicated with diabetic nephropathy, which has now become the main cause of chronic kidney disease (CKD) and end-stage renal disease. Currently, there is still a lack of specific treatment methods for diabetic nephropathy in clinical practice. Therefore, it is necessary to find new drug targets for targeted treatment of diabetic nephropathy to improve the treatment effect of diabetic nephropathy. The drug target we studied is based on the evolution process from type 2 diabetes to diabetic kidney disease, and it can not only be used during the disease progression of diabetic patients, but also be used for targeted intervention in patients with diabetic kidney disease to prevent and improve the disease progression.

[0003] The three major nutrients are interconverted and restricted to each other, and the homeostasis of amino acid and glycolipid metabolism is closely related. In the early stage of this project, amino acid mass spectrometry detection was performed on the sera of type 2 diabetes and diabetic kidney disease patients. The results showed that the glutamine and glutamine / glutamic acid ratios decreased significantly, mainly manifested as a decrease in glutamine content and an increase in glutamic acid content, indicating an increase in glutamine decomposition in the bodies of diabetic kidney disease patients, resulting in insufficient glutamine content and glutamate accumulation. Detection of the kidneys of type 2 diabetic nephropathy model mice found that the expression of kidney glutaminase (GLS1) increased significantly, resulting in a decrease in local glutamine content in the kidneys, while the level of the glutamine decomposition product glutamate increased.

[0004] CB-839 is a reversible and orally bioactive inhibitor of glutaminase 1 (GLS1), and is an orally effective inhibitor with in vivo anti-tumor activity.

[0005] In view of the significant increase in GLS1 expression in diabetic kidney disease, resulting in a decrease in local glutamine content, CB-839 was administered to inhibit the activity of GLS1, thereby maintaining the glutamine concentration in the body and inhibiting its decomposition into glutamate. Urinary biochemical results showed that the indexes of mouse renal tubular injury were significantly improved. Pathological morphology verification found that administration of CB-839 could significantly improve renal interstitial fibrosis and ectopic lipid deposition in mice.

[0006] In summary, the present invention shows that in diabetic nephropathy, the expression of glutaminase GLS1 increases, leading to a decrease in local glutamine content. By administering the GLS1 inhibitor CB-839 to inhibit its activity, endogenous glutamine decomposition is blocked, the local glutamine concentration is restored, and renal fibrosis is improved. Exogenous administration of CB-839 indicates that regulating glutamine metabolism may serve as a therapeutic target for the intervention of diabetic nephropathy, and CB-839 can be used as a therapeutic drug for improving diabetic nephropathy. Summary of the Invention

[0007] In view of the limited treatment options for diabetic kidney disease and its own metabolic characteristics, the present invention provides a pharmacological method to promote the therapeutic effect of diabetic kidney disease.

[0008] The present invention discovers a pharmacological method for treating diabetic kidney disease, which aims to supplement CB-839 exogenously to make up for the insufficient local glutamine content caused by the increased expression of GLS1 in diabetic nephropathy, treat diabetic kidney disease, delay its progression, and improve its renal function.

[0009] To achieve the above object, the present invention discloses the following technical content: The application of a small molecule compound CB-839 in the preparation of a drug for treating diabetic kidney disease. In diabetic nephropathy, the expression of glutaminase increases, resulting in increased local glutamine decomposition and decreased content. Exogenous administration of CB-839 can maintain the local glutamine content and improve diabetic nephropathy fibrosis.

[0010] The present invention discovers a method for treating diabetic kidney disease. In diabetic nephropathy, the expression of glutaminase increases and the local glutamine content decreases. CB-839 can inhibit glutamine decomposition, reduce interstitial fibrosis and ectopic lipid deposition in diabetic kidney disease, improve renal function and renal pathological structure, and achieve the effect of treating diabetic kidney disease. The small molecule compound described in the present invention has the molecular formula C26H24F3N7O3S and the English name Telaglenastat.

[0011] The positive effects of the pharmacological method for treating diabetic kidney disease disclosed by the present invention compared with the prior art are as follows: Diabetic kidney disease, as the main microvascular complication of diabetes, has an increasing incidence year by year, and its treatment options and treatment effects are limited. The present invention discovers a method for treating diabetic kidney disease. Experimental results show that CB-839 can maintain the local glutamine content in the kidney, restore the disorder of glutamine metabolism, and achieve the purpose of treating diabetic kidney disease.

[0012] The present invention is described in more detail as follows: Experimental Materials Animal models: Animal models of diabetic nephropathy: db / m mice, db / db mice; Steps

[0013] In the early stage of this project team, through amino acid metabolomics screening, it was found that compared with diabetic patients without DKD, the blood glutamine content of DKD patients decreased significantly ( Figure 1 A); compared with healthy controls, the blood glutamate content of non-DKD diabetic patients and DKD patients increased ( Figure 1 B), while the blood glutamine / glutamate ratio decreased significantly ( Figure 1 C). Correlation analysis showed that the glutamine / glutamate ratio was negatively correlated with body mass index, blood uric acid, and glucose-triglyceride index ( Figure 1 D, E, and G), while it was positively correlated with HDL-C ( Figure 1 F). Multivariate logistic regression analysis according to indicators such as age, gender, body weight, and blood lipids showed that the decrease in the glutamine / glutamate ratio was an independent risk factor for diabetes, obesity, and DKD ( Figure 1 H).

[0014] db / db mice are mice with a defect in the leptin receptor gene, showing spontaneous type 2 diabetes. Compared with db / m mice, the kidney tissues of db / db mice showed obvious pathological changes such as tubular damage, basement membrane thickening, and inflammatory infiltration. The results of Masson staining showed a significant increase in renal interstitial fibrosis in db / db mice ( Figure 2 A). By detecting the levels of glutamine and its metabolites using a kit, it was found that the glutamine content decreased significantly ( Figure 2 B), while the contents of its catabolic products glutamate and ammonia increased ( Figure 2 C, E), and the glutamine / glutamate ratio decreased significantly ( Figure 2 D). RT-PCR detected a significant increase in the content of glutamine-degrading enzymes in the kidneys of mice ( Figure 2 F). IHC and Western Blot further verified that the expression of glutamine-degrading enzymes in vivo increased ( Figure 2 G, H); these results indicated that the homeostasis of glutamine metabolism was damaged and the decomposition of glutamine in the kidneys of mice increased.

[0015] CB-839 is an inhibitor of glutaminase GLS1. After 8 weeks of exogenous intervention with CB-839 in db / db mice, it was found that CB-839 had no obvious effect on blood glucose, but could significantly reduce the body weight of mice ( Figure 3 A, B). The markers of tubular injury in the intervention group of mice decreased significantly ( Figure 3 C-E), and the local glutamine / glutamate ratio in the kidneys increased ( Figure 3F). In addition, CB-839 intervention significantly improved the pathological morphology of mouse kidney tissues, reduced interstitial fibrosis and renal interstitial lipid deposition ( Figure 3 G), and in addition, it could regulate the expression of key enzymes in renal lipid metabolism ( Figure 3 H) and reduce the deposition of renal collagen fibers ( Figure 3 I).

[0016] We found that a small molecule drug, CB-839, could alleviate the disorder of glutamine metabolism in the kidneys of db / db mice with spontaneous diabetic nephropathy model, improve renal function, relieve the damage of renal pathological structure, reduce the deposition of collagen fibers, and improve renal interstitial fibrosis. Generally speaking, our research provides new insights into the treatment of diabetic nephropathy with CB-839, and the research on CB-839 is expected to provide new targets and methods for the treatment of diabetic kidney disease. Description of the Drawings

[0018] A. Contents of glutamine in the sera of normal people, type 2 diabetes patients without nephropathy, and type 2 diabetes nephropathy patients; B. Contents of glutamate in the sera of normal people, type 2 diabetes patients without nephropathy, and type 2 diabetes nephropathy patients; C. Contents of serum glutamine / glutamate ratio in the sera of normal people, type 2 diabetes patients without nephropathy, and type 2 diabetes nephropathy patients; D. Correlation between serum glutamine / glutamate ratio and body mass index; E. Correlation between serum glutamine / glutamate ratio and uric acid level; F. Correlation between serum glutamine / glutamate ratio and high-density lipoprotein cholesterol level; G. Correlation between serum glutamine / glutamate ratio and glucose-triglyceride index; H. Serum glutamine / glutamate ratio is an independent risk factor for diabetes, obesity, and diabetic kidney disease; *: P <0.05; **: P <0.01; ***: P <0.001;

[0019] A. Staining results of the morphological structure of db / db mouse kidney tissues; B. Glutamine content in the renal cortex of db / db mice; C. Glutamate content in the renal cortex of db / db mice; D. Glutamine / glutamate ratio in the renal cortex of db / db mice; E. Ammonia content in the renal cortex of db / db mice; F. Detection of the expression of key enzymes in glutamine metabolism in the renal cortex of db / db mice by RT-PCR; G. Detection of GLS1 expression in the kidneys of db / db mice by immunohistochemistry; H. Detection of GLS1 expression in the renal cortex of db / db mice by Western blotting; *: P <0.05

[0020] A. Analysis of the body weights of mice in each group; B. Analysis of blood glucose levels in mice in each group; C. Analysis of the content of urinary NAG, a kidney injury marker, in mice in each group; D. Analysis of the content of urinary KIM-1, a kidney injury marker, in mice in each group; E. Analysis of the content of urinary β2MG, a kidney injury marker, in mice in each group; F. Changes in the glutamine / glutamate ratio in the renal cortex of mice; G. HE, PAS, MASSON, and Oil Red O staining of the kidneys of mice; H. Expression of key enzymes in lipid metabolism such as ADRP and SREBP1 in the kidneys of mice; I. Changes in the level of collagen fiber deposition in the renal cortex of mice; *: P <0.05. Detailed implementation manners

[0021] The present invention will be described below through specific implementation manners. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art. Additionally, the implementation manners should be understood as illustrative rather than limiting the scope of the present invention. The essence and scope of the present invention are only defined by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications to the material components and dosages in these implementation manners also fall within the protection scope of the present invention. The raw materials and reagents used in the present invention are all commercially available, and the small molecule compound CB-839 raw material is commercially available. Examples

[0022] We first selected 8-week-old male db / db mice as a diabetic kidney disease model. Compared with db / m mice, the expression of the glutamine-decomposing enzyme GLS1 in the kidneys of db / db mice increased, the local glutamine content in the kidneys decreased, and the glutamate content increased. Exogenous administration of CB-839 restored the local glutamine content in the kidneys ( Figure 3 F). Importantly, CB-839 significantly improved the renal function and renal pathological structure of db / db mice ( Figure 3CI), mechanistically, CB-839 significantly improved renal fibrosis and ectopic lipid deposition in db / db mice and delayed the progression of diabetic nephropathy.

[0023] The above results show that CB-839 improves glutamine metabolism in diabetic nephropathy, restores local glutamine levels in the kidney, reduces interstitial fibrosis in diabetic nephropathy, improves renal function and renal pathological structure, and achieves the effect of treating diabetic nephropathy. Example

[0024] CB-839, whose molecular structure is C 26 H 24 F3N7O3S, also known as Telaglenstat in English, is a first-of-its-kind, selective, reversible, orally active GLS1 inhibitor. CB-839 inhibits GLS1 splice variants KGA and GAC, and has higher selectivity than GLS2. CB-839 can also induce cell autophagy and has strong anti-tumor activity in animal experiments. With CB-839 as the active ingredient, pharmaceutically acceptable excipients are added to make liquid injections of various specifications according to conventional methods. There are many routes of administration for CB-839, such as injection, oral administration, etc.

[0025] (1) Preparation of injection: CB-839 200 mg, mannitol 700 mg, PEG3000 10 mg, distilled water 100 ml, adjust the pH value to 7.0, filter the filtrate to a concentration of 3 mg / ml, package it into 2 ml per ampoule, and freeze-dry to obtain the injection.

[0026] (2) Preparation of tablets: CB-839 10 mg, microcrystalline cellulose 35 mg, starch 45 mg, polyvinyl pyrrolidone 4 mg, sodium carboxymethyl starch 4.5 mg, magnesium stearate 0.5 mg, talc 1 mg; CB-839 active ingredient, starch and cellulose are sieved and fully mixed, polyvinyl pyrrolidone solution is mixed with the above powders, sieved to obtain wet granules, which are dried at 50°C, sodium carboxymethyl starch, magnesium stearate and talc are pre-sieved, and then added to the above granules for tableting.

[0027] (3) Preparation of capsules CB-839 10 mg, active ingredient excipients are sieved through 100 mesh sieves respectively, the main drug and excipients in the prescribed amount are weighed and mixed thoroughly, an appropriate amount of hydroxypropyl methylcellulose solution is added to prepare a soft material, sieved through a 24 mesh sieve, and the wet granules are dried in an oven at 60°C for about 2 hours, magnesium stearate and talc are mixed evenly with the granules, granulated, the intermediate content is determined, and filled into No. 2 capsules.

[0028] After the preferred embodiments of the detailed description, those skilled in the art can clearly understand that various changes and modifications can be made without departing from the scope and spirit of the above-mentioned patent application. Any simple modification, equivalent change, and modification made to the above embodiments based on the technical essence of the present invention all belong to the scope of the technical solution of the present invention. Moreover, the present invention is not limited by the implementation manners of the examples given in the specification.

Claims

1. Use of the small molecule compound CB-839 in the preparation of a drug for treating diabetic kidney disease; the treatment of diabetic kidney disease refers to: CB-839 significantly improves the renal function and kidney injury of diabetic kidney disease.

Images